We are investigating the proteins used to control the level of mutations produced during the replication and maintenance of genetic information in cells. For this purpose an assay has been developed to determine the frequency and specificity of mutations produced during a single round of DNA synthesis within a biologically active DNA molecule, and the accuracy with which each of the three major classes of eucaryotic DNA polymerases (Alpha, Beta, and Gamma) synthesize DNA has been determined, including sequence analysis of 1163 independent mutants. These analyses lead to the following conclusions: 1. DNA synthesis in vitro using purified eucaryotic DNA polymerases is not accurate enough to account for spontaneous mutation rates in vivo. 2. Each of the three animal cell DNA polymerases produces three major types of errors: base substitutions, frameshifts, and large deletions. For all three types of errors, the relative accuracies of the DNA polymerases are Gamma Alpha Beta. 3. Fidelity and processivity are strongly correlated. 4. Mutational specificity is influenced not only by base hydrogen bonding and stacking interactions within the DNA, but also by the protein(s) involved in the synthesis of DNA. 5. Certain single-base substitution errors may result from transient "dislocation" of a primer-template rather than from misinsertion by DNA polymerase. 6. During the course of this work, a new technique was developed for site-specific mutagenesis. It is capable of producing a desired DNA sequence alteration simply and with high efficiency, even without phenotypic selection. These experiments should provide detailed information about the protein-nucleic acid interactions which are important in determining accuracy. The studies are being expanded to employ yeast DNA polymerases (one of which is capable of proofreading) as well as "accessory" proteins which could improve fidelity.